HAL Id: hal-01607287
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Submitted on 6 Jun 2020
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Porcine SAA is not likely an apolipoprotein bound to circulating HDL3
Soler Laura, Ana Gutiérrez, P. David Eckersall, Nicola Merola, José J. Cerón, Théo Niewold
To cite this version:
Soler Laura, Ana Gutiérrez, P. David Eckersall, Nicola Merola, José J. Cerón, et al.. Porcine SAA is not likely an apolipoprotein bound to circulating HDL3. 8. European Colloquium on Acute Phase Proteins, Aug 2010, Helsinki, Finland. �hal-01607287�
“
Porcine Serum Amyloid A is not
likely an apolipoprotein bound to
circulating HDL
”
Laura Soler, Ana Gutiérrez, P. David Eckersall, Nicola
Merola, Jose J Cerón, Theo Niewold
Facultad de Veterinaria Departamento de Medicina y Cirugía AnimalVeterinary clinical analysis research group, University of Murcia
Theo Niewold
SERUM AMYLOID A (SAA)
Acute phase, 14 kDa protein, multimers
SAA1 and SAA2
Mainly produced in liver
Circulating
Apolipoproteins
Neutral IEP
SAA3
Mainly produced locally
Not circulating
Alkaline IEP
IntroductionPORCINE SAA:
THE BIG UNKNOWN
Purification of pig SAA not published
Nucleotide and amino acid sequences of each SAA
isoform has not been reported
No reports on identification by proteomic techniques
Importance: swine resistance to amyloidosis
development
IntroductionOBJECTIVES
Purifying SAA from porcine serum using traditional
protocols and study its properties
Analyzing predicted properties from sequences
deposited in UniProtKB database by using ExPASy
server sequence analysis tools
Material and Methods
3
Gradient density ultracentrifugation using
iodixanol
1
Guanidine hydrochlorideeluted HIC
2
Ethanoleluted HIC
SAA purification from pig serum
1. Rabbit antibovine SAA3 pAb Dr. Molenaar
2. Rat antiporcine recombinant SAA mAb
Search UniProtKB database for porcine SAA sequences
and analyze with ExPASy tools (ProtParam, GRAVY)
Material and MethodsSDSPAGE and immunoblotting analysis
Sequence analysis
•
Hydrophobic interaction
chromatography
70 kDa 65 kDaGuanidine hydrochloride
Ethanol
ResultsHIC G FG G HIC Et 60 kDa SAA3b 30 kDa 20 kDa 15 kDa FG G HIC Et H IC G F G G H IC E t S A A 3 b M K
Rat mAb
Rabbit
pAb
•
Immunobloting analysis
SDSPAGE
SAAp MK Results Porcine SAA binds to the HIC columns and
need chaotropic agents, like guanidine
hydrochloride, to elute.
The purification showed has a very low
throughput and yield
It seems that the bulk of the SAA remain
unbound to the HIC columns and is lost during the
process
Results from HIC chromatography:
Results from HIC chromatography:
•
Gradient Density Ultracentrifugation
Sudan
Coomassie
VLDL
LDL
HDL
Proteins
ResultsRabbit pAb
60 kDa SAA3b SAAp MK HDL HDLHDL
Proteins
Rat mAb
•
Immunobloting analysis
Results 40 kDa 28 kDa 14 kDaResults from SAAHDL isolation by gradient
Results from SAAHDL isolation by gradient
density ultracentrifugation:
density ultracentrifugation:
Porcine SAA is present in HDLrich fractions
Immunoblot analysis indicates that the bulk of
SAA in porcine serum is not bound to HDL, but
soluble and present in the serum protein layer
MKLSTGIIFCFLILGVSSQRWASFLKEAGQGAKDMW RAYSDMREANYKNSDKYFHARGNYDAAQRGPGGA WAAKVISDARENVQRVTDLFKHGDSGHGVEDSRAD QAANAWGRSGKDPNHFRPRGLPDKY Q2HXZ9 B9P414
Q2HXZ9
Chang et al., 2007,
Liver
, 130 aa, translated from
mRNA
B9P414
Rodriguez et al., 2007,
Mammary gland
, 89 aa, translated
from mRNA
Results